Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA. msaier@ucsd.edu

As a result of recent genome sequencing projects as well as detailed biochemical, molecular genetic and physiological experimentation on representative transport proteins, we have come to realize that all organisms possess an extensive but limited array of transport protein types that allow the uptake of nutrients and excretion of toxic substances. These proteins fall into phylogenetic families that presumably reflect their evolutionary histories. Some of these families are restricted to a single phylogenetic group of organisms and may have arisen recently in evolutionary time while others are found ubiquitously and may be ancient. In this study we conduct systematic phylogenetic analyses of 26 families of transport systems that either had not been characterized previously or were in need of updating. Among the families analyzed are some that are bacterial-specific, others that are eukaryotic-specific, and others that are ubiquitous. They can function by either a channel-type or a carrier-type mechanism, and in the latter case, they are frequently energized by coupling solute transport to the flux of an ion down its electrochemical gradient. We tabulate the currently sequenced members of the 26 families analyzed, describe the properties of these families, and present partial multiple alignments, signature sequences and phylogenetic trees for them all.

The book ends with a stimulating chapter from Saier &Tseng on the evolutionary origins of transport systems, withseveral surprises about the likely evolutionary origin andfrequency of `genesis' of permease families, topologicalconstraints operating in membrane transporters, and mosaicand modular construction. This topic ­ how it all started, howit may have evolved and how it may evolve in future ­ is afitting terminal chapter to the symposium volume.

The study of biofilm considers the close association of micro-organisms with each other at interfaces and the consequent physiological adaptation to the proximity of other cells and surfaces. As such it is relevant to a variety of disciplines, including medicine, dentistry, bioremediation, biofouling, water technology, engineering and food science. Although the habitats studied, and their associated cellular communities, differ widely, some common elements exist such as method of attachment, coadhesion, regulation of biofilm phenotype and biofilm architecture. This book aims to distil the common principles of biofilm physiology and growth for all interested disciplines. It will appeal to the specialist biofilm researcher as well as to students wishing to introduce themselves to the topic.

Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA.

We have conducted bioinformatic analyses of integral membrane transport proteins belonging to dozens of families. These families rarely include proteins that function in a capacity other than transport. Many transporters have arisen by intragenic duplication, triplication and quadruplication events, in which the numbers of transmembrane alpha-helical hydrophobic segments (TMSs) have increased. The elements multiplied may encode two, three, four, five, six, 10 or 12 TMSs and gave rise to proteins with four, six, seven, eight, nine, 10, 12, 20, 24 and 30 TMSs. Gene fusion, splicing, deletion and insertion events have also contributed to protein topological diversity. Amino acid substitutions have allowed membrane-embedded domains to become hydrophilic domains and vice versa. Some evidence suggests that amino acid substitutions occurring over evolutionary time may in some cases have drastically altered protein topology. The results summarized in this microreview establish the independent origins of many transporter families and allow postulation of the specific pathways taken for their appearance.

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Evolution of small integral membrane transporters from one, two or three TMS precursors As noted above, many channel proteins/peptides exhibit just one or two TMSs (Fig. 1A). We currently have no proof of an intragenic duplication event allowing a primordial single TMS protein or peptide to give rise to a two TMS protein. However, one family of two TMS proteins, the bacterial type III target cell pore (III TCP) family (TC #1.C.36), gave comparison scores in excess of 9 SD (20– 30% identity; 65–80% similarity) when the first and second TMSs were compared (X. Zhou and M. H. Saier, Jr., unpublished results). Because the regions compared were short ( < 30 residues), we do not consider that these values establish homology (Saier, 1994; 2000a). This illustrates the difficulty in evaluating statistical significance for comparisons made between very short protein sequences. Our failure to establish homology in such cases probably does not reflect the absence or rarity of such duplications during evolutionary history.

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An example of a three TMS´four TMS interconversion exists in the Mot/Exb superfamily (TC #1.A.30; Cascales et al., 2001; Table 3). MotAB serves as a proton channel that allows bacterial flagellar rotation to be coupled to the proton motive force (pmf) by catalysing transmembrane proton flux. The MotA and MotB proteins together comprise the H+ channel. MotA is a four TMS protein, whereas MotB is a one TMS protein. Homologues of the MotA–B pair include the ExbB–D pair, which together are believed to provide the proton channel that allows the pmf to drive active transport across the outer membrane of the Gramnegative bacterial envelope (Cascales et al., 2001). The three TMS ExbB protein is homologous to the four TMS MotA protein, whereas the one TMS ExbD protein is homologous to MotB. The N-terminal TMS of MotA is lacking in ExbB. Although it is impossible to know whether a three or four TMS protein was the precursor of these homologues, it is clear that three and four TMS homologues have arisen by the gain or loss of an N-terminal TMS.

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The several dozen examples that have been cited in this review provide an emerging picture of how the majority of transmembrane transport proteins probably evolved. The primordial systems were simple one, two or three TMS peptides that could form flexible oligomeric channels. These provided simple transmembrane diffusion functions with low degrees of substrate selectivity. A need for higher degrees of specificity, for coupling two or more transport processes (i.e. co-transport, antiport) and for energy coupling for solute accumulation or expulsion provided the driving force for carrier evolution. The formation of stereospecific binding sites, strict stoichiometric recognition and conformational coupling either within the transporter polypeptide chain itself or with other superimposed energy-coupling subunits were all required.

Such requirements resulted, first, in the evolutionary appearance of more constrained channels that could assume carrier functions, secondly, in the appearance of larger, more complex obligatory secondary carriers that could no longer catalyse passive diffusion and, thirdly, in the emergence of primary active transporters and group translocators with superimposed energy-coupling subunits. Thus, the ancestral precursors of all these transporter types were simple peptide channels. The pathways most frequently taken were evidently tandem intragenic duplications giving rise to larger helical bundles that had the potential to form discrete stereospecific intramembranous substrate binding sites. They could also be constrained for coupling to other transport processes and, through conformational coupling, they were subject to control by a superimposed primary energy-yielding process such as ATP hydrolysis. We assume that none of these requirements could be satisfied by the ancestral one, two or three TMS polypeptides that served as the ancestral precursor as no such carrier has yet been documented in the scientific literature. Thus, the primary advantages provided by intragenic duplication or triplication to generate large transmembrane proteins over oligomerization of smaller peptides were (i) specificity; (ii) stoichiometric transport coupling; and (iii) control by energy expenditure. These last two processes allow accumulation of specific solutes within or expulsion of solutes from a cell, processes that simple channels are incapable of catalysing.

Experimental confirmation of this proposal will require a combination of bioinformatic and molecular biological approaches. The functional consequences of the proposed structural constraints are likely to prove more difficult to ascertain than the structural constraints them-selves. Future studies are likely to shed light on such structure–function relationships, particularly as three-dimensional structures of more transport proteins become available. They should also allow more extensive documentation of the evolutionary pathways identified here and reveal new types of evolutionary processes not yet recognized. Multiple approaches should allow definition of the requirements for flexibility and rigidity in the construction and evolutionary modification of transport proteins.

Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins.